Enzymes Flashcards
salting out
when salt concentration becomes such that molecules become less soluble and will precipitate bc salt is more interested in interacting with itself
salting in
adding salt so that the molecules become more soluble, will reach a finite max point and then decrease and move into salting out
precipitation with organic solvents does what to proteins solubility?
reduces protein solubility because it reduces surface hydration ( the thin rim of moisture around a protein) and increases the hydrophobic surface which promotes aggregation just as salting out does
what is the principle effect of precipitation with organic solvents?
reduction of water activity
how do you ensure that the enzyme does not become deactivated when using solvent precipitation?
low temp
cations exchangers bind to ?
cations and then anions are eluted
anion exchangers bind to ?
anions, so cations are eluted
strong ion exchangers
shows no change in ion exchange capacity with changes in pH
stay stable’s in large pH range
weak ion exchangers
get ionized over a limited pH range. restrictive in the pH range, do not retain there charges well
example of weak ion exchanger?
carboxymethyl cellulose
example of a strong ion exchanger?
sulphuric acid
isoelectric focusing
the molecules migrate and then stop at the pH corresponding to the pI
methods to test that an enzyme is pure?
- homogeneity- one band in electrophoresis
- chromatographic behavior
- activity testing
- isoelectric testing
expand on chromatographic behaviour in testing for enzyme purity?
plot a graph with ABS on Y and fraction number on X
-
glucose oxidase can oxidize glucose into
less sweet gluconic acid
sometimes referred to as desugaring agent
what reaction is used to remove O2 from reaction mixture?
glucose oxidase
transglutaminase is used for
forming isopeptide bond bc formed between the side chains and makes molecule bigger- food texture in imitation fish
lyases
act on double bonds, to break or create
ligases
smaller to bigger molecules
glue together
how is catalysis measured?
either through product formation or substrate dissaperence
initial rate method
take measurement as close to zero as possible
- at the start you expect the enzyme to be the most active (most potent), the substrate concentration is at max and there should be no product formed
end point method
take measurement after leaving reaction to proceed for an interval of time
- sub concentration is depleted, products would affect negative feedback inhibition
enzyme could lose activity over time
which requires more skill and expensive equipment?
initial rate method
what is happening in steady state (region b)
break down and forming at the same time
irreversible inhibitors examples
PMSF
DIFP- a nerve poison
difference in binding between irreversible and reversible inhibitor?
irreversible inhibitors bind covalently
reversible bind non-covalently
wine making and brewing use? (2)
proteases and amylases
Carbs turn to _______ ( by amylases and carbohydrases
simple sugars
simple sugars turn to _______- (by yeasts)
alcohol
what is the purpose of using proteases in wine making?
they break down proteins into soluble peptides that remain in the solution even during cold storage
what is chill proofing of beer/wine? how is it achieved?
treatment to prevent the development of haziness or cloudiness due to precipitation of proteins when beer is chilled. Treatments include the addition of tannins to precipitate proteins, materials such as bentonite to adsorb them and proteolytic enzymes to hydrolyse them.
describe proteases role in baking?
break down gluten –> make dough more pliable
why add lipoxygenases to baking?
break down carotenoid pigments to cause the whitening or bleaching of flour
why add amylases to baking?
converts starch to dextrins, oligosaccharides, and the sugar maltose. Maltose provides a fermentable sugar for the yeast–> CO2 production–> loaf volume
Transglutaminases (TGase) is used in meat too?
improve texture by bringing smaller molecules together
- strong cross-linking
- uses in gelatine, fish, meat, whey, bakery etc
- tenderize/ restructure meat
- brings aa’s together
endoproteases
used to achieve more extensive changes in the structure of a protein split peptide chain in the middle -pepsin -trypsin -chymotrypsin -elastase
exoproteases
split peptide chain at the end -remove aa residues from the terminals of proteins
- used to debater peptides and proteins
- carboxypeptidases
- aminopeptidases
cellulose is linked by?
beta 1-4 links
- Cellulose is a linear polysaccharide polymer with many glucose monosaccharide units. The acetal linkage is beta which makes it different from starch.
starch is linked by?
amylose which is alpha 1,4 and amylopectin which is alpha 1,6 links
starch or cellulose more compact?
cellulose
- straight molecule
and more resistent to enzyme hydrolyses
don’t want oxygen in a reaction, use?
glucose oxidase
4 benefits of using food enzymes?
natural
specificity
activity under mild conditions
ease of control
cheese making
rennin which contains proteolytic enzymes (chymosin and pepsins) turns caseinogen into insoluble casein- curdling milk
describe milk proteins and rennins role in curdling milk
In essence, kappa casein normally keeps the majority of milk protein soluble and prevents it from spontaneously coagulating- bc negative tails keep them repelling each other
- Chymosin proteolytically cuts and inactivates kappa casein, converting it into para-kappa-casein and a smaller protein called glycomacropeptide. Para-kappa-casein does not have the ability to stabilize the micellar structure and the calcium-insoluble caseins com together and precipitate, forming a curd.
describe alchohol fermentation biochemically?
after glycolysis= pyruvate
- alcoholic fermentation, this is a two-step process
- first involves the Enzyme pyruvate decarboxylase (PDC). The pyruvate is decarboxylated to an acetaldehyde. This acetaldehyde then undergoes a reaction catalyzed by alcohol dehydrogenase to produce ethanol; this is the step in which the NAD+ is restored
in food processing what is of most interest in lipid reactions?
hydrolysis of acyl glycerides
- mostly unfavourable
- undesired odors
TG + H2O–> glycerol and FFA
when is the action of lipase desirable?
low molecular weight products- flavour
buttermilk, baked goods, roquefort cheese
important oxidoreductase reaction in food processing?
ascorbic acid oxidase polyphenol oxidase glucose oxidase catalase lipoxygenase xanthine oxidase peroxidase
ascorbic acid oxidase
It can be oxidized by losing two protons and two electrons, but normally loses only one electron at a time
industrial application of proteases
remove bitterness modify milk and whey hydrolysis of heat gluten alcohol beverages baking recovery of scrap
lesser known applications of enzymes
-remove dental plaque
-elimination of hair with —keratinase
solubilization of cold tea solids with tannates
tannases
solubilization of cold tea solids
keratinase
elimination of hair
dextranases
remove dental plaque
controlling enzymes
temp, pressure, Aw, chemicals, enzyme (killer enzyme/anti-enzyme enzymes)
whey proteins can be hydrolyzed to _______, which are less alergetic
hydrolysates
removal of plaque
compounds in toothpastes
lysozyme break cell walls of bacteria
dextrine’s break dow sugars
proteases
